The emerging role of SARS-CoV-2 nonstructural protein 1 (nsp1) in epigenetic regulation of host gene expression

Abstract Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes widespread changes in epigenetic modifications and chromatin architecture in the host cell. Recent evidence suggests that SARS-CoV-2 nonstructural protein 1 (nsp1) plays an important role in driving these changes. Previously thought to be primarily involved in host translation shutoff and cellular mRNA degradation, nsp1 has now been shown to be a truly multifunctional protein that affects host gene expression at multiple levels. The functions of nsp1 are surprisingly diverse and include not only the downregulation of cellular mRNA translation and stability, but also the inhibition of mRNA export from the nucleus, the suppression of host immune signaling, and, most recently, the epigenetic regulation of host gene expression. In this review, we first summarize the current knowledge on SARS-CoV-2-induced changes in epigenetic modifications and chromatin structure. We then focus on the role of nsp1 in epigenetic reprogramming, with a particular emphasis on the silencing of immune-related genes. Finally, we discuss potential molecular mechanisms underlying the epigenetic functions of nsp1 based on evidence from SARS-CoV-2 interactome studies.


Introduction
T he corona virus disease of 2019  is widely regarded as one of the deadliest viral diseases in human history, with more than 7 million deaths worldwide (according to WHO epidemiological data from April 2024).The underlying cause of COVID-19 is the infection with se v er e acute r espir atory syndr ome cor onavirus 2 (SARS-CoV-2), a member of the genus Betacoronavirus of the Coronaviridae family of enveloped RNA viruses (Hu et al. 2021 ).The SARS-CoV-2 genome is a linear, nonsegmented, positive-sense ss-RNA with a 5 -cap, a 5 -UTR, two lar ge, partiall y ov erla pping open r eading fr ames (ORF1a and ORF1b) encoding nonstructur al pr oteins, se v er al nested ORFs encoding structural and accessory proteins, a 3 -UTR, and a poly-A tail (V' Kovski et al. 2021, Malone et al. 2022 ).The nonstructur al pr otein nsp1 is pr oduced earl y in infection by cotranslational cleavage of the N-terminus of the ORF1a pol ypr otein (Snijder et al. 2003 ).The nsp1 amino acid sequence is well conserved between the two highly pathogenic human corona viruses , SARS-CoV and SARS-CoV -2 (Y oshimoto 2020 ).The primary function of nsp1 is to shut off host gene expression, allowing the virus to ov ercome antivir al defense mechanisms that depend on de novo protein synthesis .T he strategies by which nsp1 inhibits host gene expression are multipronged, targeting different critical cellular pathways and functions.
The first strategy is to inhibit host translation machinery and induce endonucleol ytic cleav a ge and subsequent degr adation of cellular mRNAs (Kamitani et al. 2006, 2009, Narayanan et al. 2008, Huang et al. 2011, Lokugamage et al. 2012, Tanaka et al. 2012, Finkel et al. 2021, Lapointe et al. 2021, Abae v a et al. 2023 ).A combination of cryo-EM and biochemical studies showed that nsp1 interacts with the ribosomal 40S subunit, blocking the mRNA entry c hannel (Sc hubert et al. 2020, Thoms et al. 2020, Yuan et al. 2020 ).The replacement of two positiv el y c har ged amino acids, l ysine 164 and histidine 165, in the C-terminal region of nsp1 with alanines resulted in the loss of binding to the 40S subunit and completely abolished the nsp1-mediated host gene shutoff (Narayanan et al. 2008, 2015, Tanaka et al. 2012 ).These r esidues ar e conserv ed in nsp1 proteins from SARS-CoV and SARS-CoV-2 (Simeoni et al. 2021 ), underscoring their functional importance.To induce cellular mRNA cleav a ge and subsequent degr adation, nsp1 acts as a manganese-and calcium-dependent endonuclease e v en in the absence of any auxiliary factors (Salgueiro et al. 2024 ).Ho w ever, binding of nsp1 to the ribosome stimulates endon uclease acti vity (Tardivat et al. 2023 ).Interestingly, a conserved SL1 hairpin found at the 5 end of viral genomic and subgenomic RNAs (Miao et al. 2021, Bujanic et al. 2022, Sosnowski et al. 2022 ) protects them from nsp1-mediated cleav a ge and degr adation (Finkel et al. 2021, Tardivat et al. 2023 ), suggesting a mechanism by which viral genes escape global translational shutoff.
The second strategy used by nsp1 to suppress host gene expression is to block the nuclear export of cellular mRNAs .T his is ac hie v ed independentl y of nsp1 binding to the ribosome (Fisher et al. 2022 ) through a direct interaction between nsp1 and the mRNA n uclear export rece ptor NXF1 (Zhang et al. 2021a ).Furthermore, nsp1 binding to nucleoporins (Gomez et al. 2019, Zhang et al. 2021a ), whic h ar e the building bloc ks of the nuclear por e complex (NPC) (Kim et al. 2018, Yang et al. 2023 ), may alter the NPC structure and interfere with NXF1-mRNA docking at the NPC.The resulting nsp1-mediated inhibition of mRNA nuclear export reduces antivir al pr otein synthesis while incr easing the av ailability of the host cell translation machinery to viral RNAs.
The third strategy by which nsp1 affects host gene expression is by antagonizing type I interferon (IFN-I) signaling, whic h pr omotes the activ ation of antivir al interfer on-stim ulated genes (ISGs).The expression of these genes is controlled by the JAK/ST A T signaling pathway, which involves the phosphorylation of ST A T1/ST A T2 transcription factors to facilitate their nuclear translocation in a complex with a third transcription factor, IRF9 (Platanias 2005, Au-Yeung et al. 2013 ).Ther efor e, interfering with ST A T phosphorylation provides a possible means for the virus to suppr ess ISG expr ession in infected cells.Indeed, nsp1 has been shown to inhibit the phosphorylation of ST A T1 in cells stimulated with IFN-α (Wathelet et al. 2007, Xia et al. 2020 ).T hus , nsp1 has the ability to specificall y bloc k ISG expr ession by inhibiting the JAK/ST A T signaling pathway at the le v el of ST A T1 phosphorylation.Furthermore, nsp1 inhibits the upstream retinoic acidinducible gene I (RIG-I) pathway, which connects the RIG-I-like rece ptor, a k e y sensor of viral infection, to type I interferon transcription (Xia et al. 2020 ).Taken together, the above findings indicate that nsp1 is a sur prisingl y m ultifunctional pr otein that helps the virus e v ade host defenses by interfering with various cellular processes and pathwa ys .Howe v er, r ecent e vidence suggests that, in addition to these functions, nsp1 is also dir ectl y involv ed in epigenetic regulation.
This r e vie w does not attempt to cov er all aspects of how nsp1 affects host mRNA translation, stability, nuclear export, and innate immune responses, as these have already been thoroughl y r e vie wed else wher e (Yuan et al. 2021, Kar ousis 2024 ).Instead, we will focus on the emerging role of nsp1 in epigenetic regulation.We will first briefly outline how SARS-CoV-2 infection alters histone post-translational modifications (PTMs) and c hr omatin arc hitectur e in the host cell.We will then summarize the k e y findings supporting the central role of nsp1 in epigenetic r epr ogr amming during SARS-CoV-2 infection.Finally, we will discuss potential molecular mechanisms underlying nsp1-mediated epigenetic silencing of antiviral immune-related genes.

SARS-CoV-2 infection interferes with DNA and histone epigenetic modifications
Epigenetic modifications of nucleic acids (Chen et al. 2017a ) and histones (Zhang et al. 2021b ) allow the cell to quic kl y r espond to v arious envir onmental and pathogenic stim uli by r epr ogr amming gene transcription (Chen et al. 2017b, Zhang et al. 2020b, Fritz et al. 2022 ).While the cellular mechanisms underlying such epigenetic modifications play an important role in antiviral defense, viruses can also hijack them for their own benefit (Tsai and Cullen 2020 ).A growing body of evidence suggests that SARS-CoV-2 infection affects both DNA methylation and histone PTMs, which are the two fundamental epigenetic mechanisms that regulate gene expression.
Se v er al studies have used circulating blood cells from COVID-19 patients to investigate how SARS-CoV-2 infection affects DNA methylation, an epigenetic modification associated with gene silencing (Newell-Price et al. 2000 ).Although blood cells are not the primary target of SARS-CoV-2, the virus has been shown to infect monocytes (Codo et al. 2020, Pontelli et al. 2022 ), monocytederiv ed macr opha ges, dendritic cells (Zheng et al. 2021 ), T lymphocytes (Pontelli et al. 2022, Shen et al. 2022 ), and B lymphocytes (Pontelli et al. 2022 ).Depending on the blood cell type, the infection can be either pr oductiv e or abortiv e , angiotensin-con verting enzyme 2 (ACE2)-dependent or independent.Corley et al. ( 2021 ) identified m ultiple differ entiall y methylated loci in the peripheral blood mononuclear cells (PBMCs) of se v er e COVID-19 patients .T hey sho w ed that the DN A methylation signature associated with se v er e COVID-19 is c har acterized by h ypermeth ylation of IFN-related genes and h ypometh ylation of inflammatory genes, supporting the hypothesis that SARS-CoV-2 hijacks the host epigenome to suppress antiviral immune responses and promote uncontrolled inflammation.In addition, the researchers discov er ed that DNA methylation aging clocks were accelerated in se v er e COVID-19 patients.Zhou et al. ( 2021 ) investigated genomewide DNA methylation profiles in the whole blood of healthy subjects and COVID-19 patients with differ ent le v els of disease se v erity.They found that the SARS-CoV-2 infection caused a global repr ogr amming of DNA methylation in blood cells of infected patients .T he authors linked the changes in DNA methylation to the regulation of inflammatory and imm une-r elated genes, further supporting a clinically relevant role for this epigenetic mechanism.Other studies have also linked the differ entiall y methylated DNA regions in peripheral blood cells of COVID-19 patients to genes involved in antiviral immune responses, leukocyte activity, and autoimmune diseases (Balnis et al. 2021, Castro de Mour a et al. 2021, Konigsber g et al. 2021, Bartur en et al. 2022 ).Using a mouse model of SARS-CoV-2 infection, Li et al. ( 2021a ) identified multiple differentially methylated sites at gene promoters in the heart and kidney of infected animals.Notably, both organs exhibit SARS-CoV-2 tropism in humans (Liu et al. 2021 ).The study by Noguera-Castells et al. ( 2024 ) stands out for its focus on altered DNA methylation in the lung, which is the primary target of SARS-CoV-2 infection.The authors analysed lung autopsy samples from patients who died of COVID-19-related pneumonia to identify the DNA methylation signature associated with r espir atory failur e .T he identified signature was enriched in genes related to inflammation, cell adhesion, and imm une r esponse, suggesting the clinical significance of epigenetic changes in DNA methylation during native SARS-CoV-2 lung infection.These r esults ar e particularl y important because they show that SARS-CoV-2 has a universal effect on DNA methylation signatures not only in peripheral blood cells but also in lung epithelial cells, the primary target of infection.Finally, SARS-CoV-2 infection leaves long-term epigenetic traces in PBMCs or total leukocytes of recovered patients in the form of altered DNA methylation patterns (Balnis et al. 2022, Huoman et al. 2022, Nikesjo et al. 2022 ), suggesting a role for this modification in the persistence of postacute sequelae of COVID-19, also known as "long CO VID." PTM of histones is another k e y e pigenetic mechanism that r egulates gene expr ession.Ther e is gr owing e vidence that SARS-CoV-2 infection has a profound effect on histone PTMs.Histone H3 lysine 27 acetylation (H3K27ac), a PTM associated with active enhancers, is one notable example.Wang et al. ( 2023 ) sho w ed that infection of cultured ACE2-overexpressing A549 lung cells (A549-ACE2) with SARS-CoV-2 caused a global decrease in this activ e c hr omatin mark, whic h was associated with tr anscriptional r epr ession of host genes involved in the antivir al imm une r e-sponse.At the same time, the authors found a specific increase in the activ e pr omoter mark histone 3 lysine 4 trimethylation (H3K4me3) at proinflammatory gene promoters, suggesting an epigenetic mechanism for the activation of proinflammatory cytokines observed in COVID-19 patients.Yang et al. ( 2022 ) analysed the profiles of H3K4me3 and the r epr essiv e mark histone 3 lysine 27 trimethylation (H3K27me3) in PBMCs from hospitalized COVID-19 patients .T he y found selecti v e c hanges in H3K4me3 or H3K27me3 near the transcription start sites of se v er al micr oRNAs that r egulate imm une and inflammatory r esponses.Le ppk es et al. ( 2020) sho w ed that blood serum of COVID-19 patients had incr eased le v els of another epigenetic modification, citrullination of histone 3 (Cit-H3).They found that the increase in Cit-H3, an epigenetic mark of decondensed and tr anscriptionall y activ e c hr omatin (Christophorou et al. 2014 ), was associated with increased proinflammatory cytokine expression and release of extracellular c hr omatin tr a ps (NETs) by neutr ophils during se v er e COVID-19.Yet another epigenetic PTM affected by SARS-CoV-2 infection is histone pr oteol ytic cleav a ge.Histone cleav a ge is an emerging epigenetic mechanism whose role in regulating chromatin structure and gene expression is only beginning to be understood (Yi and Kim 2018 ).Huckriede et al. ( 2021 ) detected extracellular histone H3 in the plasma of hospitalized se v er e COVID-19 patients and sho w ed that the H3 was often pr oteol yticall y cleav ed.The authors hypothesized that the cleav a ge, most likel y performed by neutrophil elastase at the N-terminal tail of H3, may be part of a yet-to-be-described NETosis-related regulatory mechanism.Although many of the above studies are limited by the use of in vitro -cultured lung cells or peripheral blood cells, which are not the primary target of SARS-CoV-2, there is a growing consensus that epigenetic modifications of DNA and histones play an important and clinically relevant role during SARS-CoV-2 infection.Nonetheless, r esearc h on cor onavirus-induced epigenetic modifications is still in its early stages , lea ving many questions unans wered.For example , the molecular mec hanism(s) underl ying the locus-specificity of virus-induced epigenetic modifications remain lar gel y unknown.Without this knowledge, it would be difficult to understand how SARS-CoV-2 epigenetically silences antiviral imm une r esponse genes while activ ating others, suc h as those involved in inflammation.For information on enzymes, which may be involved in epigenetic modifications in the context of SARS-CoV-2 infection, we refer the reader to comprehensive reviews av ailable else wher e (Kgatle et al. 2021, Bhat et al. 2022, Foolc hand et al. 2022, Dey et al. 2023 ).

SARS-CoV-2 infection rearranges host chromatin architecture
Dynamic changes in chromatin architecture play an important role in gene regulation during development, normal physiology, and disease (Pombo and Dillon 2015 ).Se v er al viruses are known to interfere with epigenetic mechanisms that regulate host chromatin arc hitectur e to establish persistent infection and e v ade host defenses (Tsai andCullen 2020 , Friedman et al. 2022 ).One such virus is SARS-CoV-2.Ho et al. ( 2021 ) used the Hi-C c hr omosome conformation ca ptur e tec hnique to demonstr ate that SARS-CoV-2 infection causes extensive rearrangement of chromatin topology in cultured A549-ACE2 cells .T hey sho w ed that the infection caused a significant redistribution of active (A) and inactive (B) c hr omatin compartments in infected cells .T his redistribution was c har acterized by the shortening of long A and B c hr omatin domains and their conversion into mixed A-B subdomains.In addition, the authors performed c hr omatin imm unopr ecipitation and sequencing (ChIP-seq) analysis for H3K27ac, an epigenetic mark of active enhancers, to show the association of SARS-CoV-2induced c hr omatin r estructuring with c hanges in tr anscriptional activity.These changes affected genes involved in proinflammatory and antivir al imm une r esponses, suggesting that they are the targets of virus-induced epigenetic reprogramming.A more recent study by Wang et al. ( 2023 ) found that SARS-CoV-2 infection globall y r estructur es host c hr omatin in A549-ACE2 cells b y w eakening compartment A, A-B mixing, and reducing contacts within self-inter acting topologicall y associating domains (TADs).Importantly, genomic loci containing ISGs had fewer intra-TAD promoter-enhancer contacts and less H3K27ac deposition in SARS-CoV-2-infected cells compared to mock-infected controls.The findings of Wang et al. ( 2023 ) were recently confirmed in a study employing polymer physics-based molecular modeling.The study sho w ed that SARS-CoV-2 infection caused TAD r earr angements at ISG genomic loci by reducing the specificity and structural stability of regulatory contacts (Chiariello et al. 2023 ).In addition, se v er al independent studies have found that SARS-CoV-2 induces large-scale chromatin remodeling in COVID-19 patients' PBMCs or sorted CD14 + monocytes, affecting the expression of imm une pathway-r elated genes.Suc h c hr omatin r emodeling was influenced by the stage and severity of the disease, as well as the age of the patient (Zheng et al. 2020, Li et al. 2021b, Brauns et al. 2022, Giroux et al. 2022 ).Furthermore, SARS-CoV-2 infection induced extensive and long-lasting changes in chromatin accessibility and transcriptional profiles of convalescent COVID-19 patients (You et al. 2021, Cheong et al. 2023 ).T hese changes ma y r epr esent an epigenetic memory of a pr e vious imm une c hallenge by SARS-CoV-2 that pr ogen y monocytes inherit from their progenitors (Cheong et al. 2023 ).Ho w e v er, this epigenetic imm une memory, also known as trained immunity, may not only protect against reinfection but also contribute to the long-term clinical sequelae of COVID-19.Taken together, these findings suggest that epigenetic mechanisms involving genome-wide c hr omatin r emodeling contribute to various immune and inflammatory phenomena observed in COVID-19 patients.We are making gradual progress in understanding these mechanisms, but there are still many unans wered questions .Among these questions , one is particularly important: whic h vir al factors ar e involv ed in these epigenetic mec hanisms?
Unveiling the role of nsp1 as an epigenetic regulator Lee et al. ( 2023 ) emplo y ed Assay for Transposase-Accessible Chromatin using sequencing (A T AC-seq) to assess genome-wide c hanges in c hr omatin accessibility in cultur ed Ver oE6 cells tr ansientl y tr ansfected with plasmids encoding all SARS-CoV-2 pr oteins (Lee et al. 2023 ).Of the 29 proteins tested, only two, nsp1 and, some what unexpectedl y, spike, induced widespr ead c hanges in c hr omatin accessibility.The fact that nsp1 and spike, e v en when expr essed alone, wer e able to globally modify the c hr omatin accessibility landscape suggested an active role for these proteins in genome-wide c hr omatin r estructuring.Ho w e v er, the authors found significant differences between the chromatin accessibility profiles of nsp1-and spike-expressing cells.First, nsp1 had a more global effect on c hr omatin accessibility, with at least twice as many A T AC-seq peaks induced by nsp1 compared to those induced by spike .T he n umber of both positi ve and negati ve A T ACseq peaks was higher in nsp1-expressing cells, indicating that nsp1 generated more regions with increased and decreased chromatin accessibility than spike.Second, and most importantly, the c hr omatin accessibility signatur es of nsp1 and spike were significantl y differ ent, with onl y a minor ov erla p.One explanation for this is that nsp1 promotes a different transcriptional program than spike, possibly through a different mechanism.Furthermore, the authors sho w ed a significant and positive correlation between c hanges in c hr omatin accessibility induced by nsp1 and those observed during SARS-CoV-2 infection, confirming that A T AC-seq data obtained with individuall y expr essed nsp1 is physiologically r ele v ant.
The simplest and most straightforw ar d explanation for the widespread nsp1-induced changes in chromatin accessibility is that nsp1, a known translational repressor, inhibits the translation of mRNAs encoding master transcriptional and epigenetic regulators.Ho w ever, a recent study by Anastasakis et al. ( 2024 ) provides another explanation: the existence of an nsp1-mediated, translation-inde pendent e pigenetic mechanism.The stud y combined se v er al high-thr oughput a ppr oac hes to inv estigate how nsp1 affects the expression of host imm une-r elated genes.First, the authors used RNA-seq in tr ansientl y tr ansfected A549 cells to show that nsp1 expression significantly reduced the steady-state mRNA le v els of host genes involved in innate immunity and antiviral defense .T he anal ysis was performed within a narr ow time window of 16-24 hours post-transfection when nsp1 expression le v els wer e still low, mimic king the situation earl y in SARS-CoV-2 infection.Within this time window, nsp1 expression levels are still insufficient to suppress host cell mRNA translation through stoichiometric binding to the host translation machinery.To test this experimentally, Anastasakis et al. ( 2024 ) used Ribo-seq, also known as ribosome profiling, to determine how nsp1 expression affects the efficiency of host gene translation.They found that earl y after tr ansfection, nsp1 had no effect on the translation efficiency of the downregulated genes identified by RNA-seq, implying that inhibition of translation could not explain the reduced expr ession of antivir al imm une-r elated genes .T his prompted the authors to investigate whether nsp1 regulates the process of host gene tr anscription itself, r ather than tr anslation or stability of the r esulting tr anscripts .T hey performed RNA pol ymer ase II (Pol II) ChIP-seq and found that nsp1-induced changes in Pol II occupanc y correlated w ell with changes in mRNA le v els fr om RNAseq data.Furthermore, they sho w ed that nsp1 increased the deposition of the r epr essiv e histone H3 lysine 9 dimethylation mark (H3K9me2) at the silenced imm une-r elated genomic loci.Finally, they demonstrated that specific pharmacological inhibition of the H3K9me2 methyltr ansfer ase G9a, alternativ el y known as euc hr omatic histone l ysine N -methyltr ansfer ase 2, r estor ed the expr ession of antivir al imm une-r elated genes suppr essed by nsp1 and significantly inhibited SARS-CoV-2 infection, resulting in an ∼10-fold reduction in viral load.T hus , the results of Anastasakis et al. ( 2024 ) suggest that nsp1 inhibits host antiviral defenses not only at the cellular mRNA le v el, but also at the epigenetic le v el.Specificall y, nsp1 induces epigenetic silencing of immunerelated genes through the G9a-mediated deposition of the signatur e heter oc hr omatin mark H3K9me2 at the corr esponding genomic loci.

Putati v e mec hanisms of nsp1-mediated epigenetic regulation
The molecular mechanism by which nsp1 promotes re pressi ve histone methylation to silence imm une-r elated genes is curr entl y unkno wn.Ho w e v er, the identification of host factors interacting with nsp1 suggests se v er al possibilities, whic h ar e discussed below.

Direct nsp1 association with chromatin
One possible mechanism by which nsp1 may induce epigenetic repr ogr amming is by entering the nucleus and interacting directly with the c hr omatin r emodeling mac hinery.Indeed, se v er al host proteins identified as nsp1 interactors in high-throughput studies are known to be associated with c hr omatin (Li et al. 2023 ).Although SARS-CoV-2 nsp1 is a pr edominantl y cytoplasmic protein, se v er al independent studies have shown that it also localizes to the nucleus in plasmid-transfected cells (Gordon et al. 2020a, Zhang et al. 2020a, 2021a, Lee et al. 2021, Shemesh et al. 2021 ).According to our unpublished observations, the nuclear-enriched fraction of 293T cells contains up to 10% of total cellular SARS-CoV-2 nsp1 at later time points after transfection.In SARS-CoVinfected cells, a small but distinct proportion of nsp1 is detected in the nucleus from 9 hours postinfection (Prentice et al. 2004 ).Since SARS-CoV nsp1 has more than 90% sequence similarity with its SARS-CoV-2 ortholog (Yoshimoto 2020 ), these two proteins may share a common subcellular localization.Ho w e v er, other studies have found that SARS-CoV-2 nsp1 is almost exclusiv el y located in the cytoplasm of infected cells (Gordon et al. 2020a, Shi et al. 2022 ).In any case, the functional significance of nsp1nuclear localization remains unknown.Although this seems to be an attr activ e possibility, ther e ar e no published observ ations to support the hypothesis that nsp1 is dir ectl y bound to nucleosomes .T hus , whether nsp1 exerts its epigenetic function through the direct interaction with chromatin-associated machinery remains, so far, uncertain.

Nsp1 interaction with PRRC2B
In an alternative mechanism to association with chromatin, nsp1 may act indir ectl y to induce epigenetic silencing of immunerelated genes by interacting with specific regulatory proteins.Nsp1 binding to such regulatory proteins may take place in the cytoplasm, ther eby ob viating the need for nsp1 to enter the nucleus.Anastasakis et al. ( 2024 ) focused on one such protein, PRRC2B, known to localize to both nucleus and cytoplasm (Thul et al. 2017, Jiang et al. 2023 ) and to interact with nsp1 (Samav arc hi-Tehr ani et al. 2020 ).Importantl y, the nsp1-inactiv ating double m utation K164A/H165A reduced its affinity to PRRC2B (Samav arc hi-Tehr ani et al. 2020 ), suggesting a functional role for this pr otein-pr otein interaction.What makes PRRC2B special among other nsp1 interactors is that it also interacts with the H3K9me2 writer G9a (Rual et al. 2005 ).Consistent with a potential role for PRRC2B in regulating r epr essiv e histone methylation through binding to G9a, Anastasakis et al. ( 2024) sho w ed that siRN A-mediated knockdo wn of PRRC2B r e v ersed the nsp1-induced silencing of imm une-r elated genes.Taken together, these results suggest that the mechanism of nsp1-mediated epigenetic silencing of imm une-r elated genes ma y in volve PRRC2B.Further experiments ar e r equir ed to v alidate this proposed mechanism.

Nsp1 interaction with Pol α
Another candidate mechanism for the nsp1-mediated epigenetic gene silencing involves the interaction nsp1 with DNA pol ymer ase alpha, also known as P ol α.T his four-subunit enzyme is best known for synthesizing a short RNA primer and extending it by ∼20 deo xyribon ucleotides during DNA re plication (Pellegrini 2012 ).Ho w e v er, Pol α also performs other functions, as discussed below.The four subunits of Pol α (POLA1, POLA2, PRIM1, and PRIM2), collectiv el y r eferr ed to as the primosome, wer e originally identified as nsp1 binding partners in a high-throughput proteomic study by Gordon et al. ( 2020b ).The association of nsp1 with Pol α subunits has since been confirmed in many other studies of the SARS-CoV-2-host interactome (Li et al. 2023 ).The consistent identification of Pol α as a binding partner of nsp1 prompted Kilkenny et al. ( 2022 ) to solve the cryo-EM structure of this multipr otein complex.The structur e r e v ealed that nsp1 interacts with Pol α through the inacti ve exon uclease domain of the POLA1 catalytic subunit.Consistent with this observation, the authors sho w ed that binding to nsp1 does not affect the ability of Pol α to synthesize primers for DNA r eplication.Furthermor e, the interaction between nsp1 and Pol α involves the conserved middle domain of nsp1, but not the C-terminal region responsible for ribosome targeting and inhibition of mRNA translation (Schubert et al. 2020, Thoms et al. 2020, Lapointe et al. 2021 ).T hus , the results of Kilkenny et al. ( 2022 ) suggest that the interaction between nsp1 and Pol α is unlikely to affect either host mRNA translation or DNA replication.
In addition to its role in DNA replication, Pol α has been implicated in heter oc hr omatin maintenance (Li and Zhang 2012 ) and modulation of the interferon response (Starokadomskyy et al. 2021 ).Before discussing these functions in more detail, it is important to note that all Pol α subunits ar e conserv ed acr oss eukaryotes (Shultz et al. 2007 ), suggesting a shared function in div erse or ganisms.Se v er al lines of e vidence indicate that Pol α is involved in epigenetic inheritance of heterochromatin during DNA re plication-coupled n ucleosome reassembly.Studies in fission yeast found that mutations in the catalytic subunit of Pol α r educed the r ecruitment of Swi6, a yeast ortholog of the animal heter oc hr omatin pr otein 1 (HP1), to heter oc hr omatic loci, r esulting in their activation (Ahmed et al. 2001, Nakayama et al. 2001 ).In addition, these studies sho w ed that Swi6 dir ectl y interacts with the C-terminal part of the catalytic subunit of Pol α.Similar r esults wer e obtained in plants, wher e the catal ytic subunit of Pol α was found to physically and geneticall y inter act with the Swi6 plant ortholog LHP1 (like HP1) (Barr er o et al. 2007 ).The primary function of HP1 and its orthologs is to specifically recognize the r epr essiv e H3K9me2/3 marks and promote epigenetic gene silencing through heterochromatin formation (Bannister et al. 2001, Lachner et al. 2001 ).Ho w ever, in addition to being an H3K9me2/3 reader, HP1 maintains the protein stability of H3K9 methyltransferases, including G9a, by tethering them to c hr omatin (Maeda and Tac hibana 2022 ).Consequentl y, the r ecruitment of HP1 by Pol α during DNA replication may promote the deposition of repressive H3K9 methylation marks on newly assembled nucleosomes.Consistent with this possibility, a mutation in the catalytic subunit of Pol α r educed pr omoter-associated H3K9me2 le v els in Arabidopsis (Liu et al. 2010 ).T hus , it can be hypothesized that nsp1 r epr esses the tr anscription of host imm une-r elated genes, at least in part, by exploiting the physiological mechanism that propagates the silenced c hr omatin state during host cell division.This mechanism ma y in volve the interaction of nsp1 with Pol α and oper ate thr ough the P ol α-HP1-G9a-H3K9me2 axis .T he presence of Pol α in both the nucleus and cytoplasm (Brown et al. 1981, Thul et al. 2017 ) suggests that the interaction of nsp1 with Pol α may occur not only in the nuclear compartment but also in the cytoplasm, eliminating the strict r equir ement for nsp1 to enter the nucleus.
Given the role of Pol α in heterochromatin inheritance, it is perhaps not surprising that the N-terminal region of POLA1 contains a conserved histone-binding motif.This motif interacts with histones H2A-H2B (Evrin et al. 2018 ) and H3-H4 (Li et al. 2020 ) to maintain transcriptional silencing at heter oc hr omatic r egions and facilitate the transfer of parental histones to lagging DNA strands.In addition, the nearby CIP-box sequence interacts with the trimeric scaffold protein AND-1/CTF4 (Simon et al. 2014 ), which is dispensable for DNA replication but is also involved in parental histone transfer (Evrin et al. 2018, Gan et al. 2018 ).At least five phosphorylation sites, four of which are proline-directed, were identified immediately downstream of the CIP-box sequence (Hornbeck et al. 2015 ), suggesting that phosphorylation-induced conformational changes (Wulf et al. 2005, Gurung et al. 2023 ) could regulate POLA1-mediated epigenetic mechanisms.Another regulatory hotspot in POLA1 is the first glycine residue in the conserv ed GPCWL motif, whic h is critical for binding to the SPT16 subunit of the histone c ha per one FACT (Zhou and Wang 2004 ).Notably, most of the above sequences are located in the N-terminal r egion of POLA1, whic h lac ks a defined secondary structure .T his flexible region sits immediately upstream of the catalytically inacti ve POLA1 n uclease domain, which contains the nsp1 binding site (Fig. 1 A).Ther efor e, it can be hypothesized that nsp1 promotes POLA1-mediated epigenetic silencing by pr e v enting r egulatory proteins, including enzymes involved in PTMs, from accessing their binding sites in the flexible N-terminal region of POLA1.Consistent with this possibility, we found a possible steric clash between POLA1-bound nsp1 and the flexible N-terminus of POLA1 in the cryo-EM structure published by Kilkenny et al. ( 2022 ) (Fig. 1 B).
Inter estingl y, SARS-CoV-2 infection upr egulated all four subunits of Pol α at both the mRNA and pr otein le v els (Pur a y-Cha vez et al. 2021 ), suggesting the existence of a feedback mechanism that enhances the effects of Pol α during infection.Considering the role of POLA1 in epigenetic gene silencing through the interaction with HP1 and deposition of H3K9me2, it is tempting to speculate that SARS-CoV-2-induced ov er expr ession of Pol α could pr omote e pigenetic re pression of antiviral immune-related genes in infected cells.Support for this possibility comes from research on X-linked reticulate pigmentary disorder (XLPDR), an orphan genetic disease caused by mis-splicing mutations in the POLA1 gene resulting in partial POLA1 protein deficiency (Starokadomskyy et al. 2021 ).The reduced abundance of normally spliced POLA1 transcripts causes a dramatic increase in the expression of ISGs (Starokadomskyy et al. 2019 ), leading to the de v elopment of an autoinflammatory disease in XLPDR patients .T hese findings suggest that sufficient availability of functional Pol α is essential for the tight transcriptional control of ISGs.Such transcriptional control can be brought about through an epigenetic mechanism involving r epr essiv e histone methylation and heter oc hr omatin formation.An alternative, but not mutually exclusive, mechanism ma y in volv e dampening of antivir al nucleic acid sensors by Pol α-synthesized cytosolic RNA/DNA hybrids (Starokadomskyy et al. 2016 ).T hus , genetic e vidence fr om human disease suggests that Pol α-mediated mechanism(s) control the expression of antiviral immune-related genes under normal physiological conditions.During SARS-CoV-2 infection, nsp1 may exploit these mechanisms to prevent immune-related genes from activating.One w ay ho w this can be ac hie v ed is thr ough the dir ect interaction between nsp1 and Pol α, leading to the deposition of r epr essiv e H3K9me2 marks at the corresponding genomic loci and the formation of heter oc hr omatin.Whether this speculativ e epigenetic mec hanism actuall y exists, ho w e v er, awaits further study.A complementary possibility is that nsp1 silences immunerelated genes by interfering with the Pol α-mediated synthesis of immunogenic RN A/DN A hybrids in the c ytoplasm.Ho w ever, this scenario is less likely because the primer synthesis activity of Pol α is not affected by its interaction with nsp1 (Kilkenny et al. 2022 ).The contact area between POLA1 and nsp1 is highlighted with the dashed line.Nsp1 is drawn as ribbons, with POLA1-interacting structural elements in purple and the rest in green.The dotted circle indicates a potential steric clash between nsp1 and binding partners of the flexible N-terminal region of POLA1.The schematic rendering of the nsp1-POLA1 complex is based on the published cryo-EM structure by Kilkenny et al. ( 2022 ).

Synergism between nsp1-mediated inhibition of ST A T phosphorylation and deposition of H3K9me2 on chromatin
As mentioned abo ve , nsp1 can repress ISG expression by inhibiting the phosphorylation of the transcription factor ST A T1 in the cytoplasm.Reduced phosphorylation pr e v ents ST A T1 from entering the nucleus, recognizing specific enhancers, and activating ISG transcription.As w e no w kno w, the second w ay ho w nsp1 promotes ISG silencing is by adding the r epr essiv e mark H3K9me2 at the corresponding genomic loci (Fig. 2 A).Since H3K9me2 pr e v ents gene expression by reducing chromatin accessibility to transcription factors (Padeken et al. 2022 ), these two nsp1-mediated mech-anisms are likely to be synergistic rather than mutually exclusive.cleus of unstimulated cells .T hese nuclear uST A T proteins are involved in noncanonical ST A T functions, which include basal transcriptional r epr ession (Awasthi et al. 2021 ).All ST A T transcription factors contain the conserved PxVxL/I motif in their DNAbinding domain (Fig. 2 B).This motif is specificall y r ecognized by the c hr omo shadow domain of the HP1 protein (Smothers and Henik off 2000, Lec hner et al. 2005, Br o w er-Toland et al. 2007, Xu et al. 2023 ).Since HP1 is the k e y structural component of heter oc hr omatin and the reader of H3K9me2/3 (Canzio et al. 2014 ), its interaction with nuclear uST A Ts promotes heterochromatin stability (Majoros et al. 2017 ).Although this phenomenon is best studied for uST A T3 (Dutta et al. 2020 ), uST A T5A (Hu et al. 2013 ), and Drosophila uST A T92E (Shi et al. 2008 ), there is evidence that uST A T1 also acts as a transcriptional repressor of specific genes in unstimulated cells (Zimmerman et al. 2012 ).Consistent with the role of nuclear uST A T in maintaining heter oc hr omatin stability, activation of the canonical JAK/ST A T pathway shifts the balance tow ar d pST A T, causing heter oc hr omatin disruption (Shi et al. 2006, Li 2008 ).Similarly, the ability of nsp1 to inhibit ST A T1 phosphorylation and thus pr e v ent the activ ation of the canonical JAK/ST A T pathwa y ma y tip the balance in favor of heter oc hr omatin stabilization (Fig. 2 A).Suc h heter oc hr omatin stabilization could be mediated, for example, by nuclear uST A T3.Although ST A T3 is activated by IFN-I stimulation in a manner similar to ST A T1 and ST A T2, it has the opposite (inhibitory) effect on the canonical IFN-I response by sequestering activated ST A T1 into ST A T1:ST A T3 heterodimers (Ho andIvashkiv 2006 , Tsai et al. 2019 ).Consistent with the role of ST A T3 as a suppressor of the IFN-I response, nuclear uST A T3 pr omotes heter oc hr omatin formation by binding to HP1 (Dutta et al. 2020 ).Nsp1 could stimulate this process by inhibiting ST A T1 phosphorylation, effectiv el y bloc king the canonical JAK/ST A T pathway, and thus pr e v enting ISG tr anscriptional acti vation.Collecti vely, the above studies suggest that nsp1 employs se v er al distinct but ov erla pping tr anscriptional and epigenetic strategies to effectively silence the expression of antiviral imm une-r elated genes .T hese str ategies ar e likel y to be syner gistic and involve viral interference with histone and transcription factor PTMs.

Nsp1 sequence v aria tion and epigenetic regulation
Although nsp1 is one of the least conserv ed nonstructur al pr oteins among coronaviruses (Maurina et al. 2023 ), it is highly conserved among SARS-CoV-2 variants (Ghaleh et al. 2023 , Kandwal andFayne 2023 ).This suggests that the SARS-CoV-2 nsp1 sequence is well optimized, with a low tolerance for amino acid substitutions.On the other hand, the low sequence similarity of nsp1 among coronaviruses indicates that it is a hotspot of genetic variation, which allows the virus to adapt to different hosts, cell types, tissues , and other en vironmental factors .One possible strategy for the virus to ac hie v e suc h ada ptation is to selectiv el y modify host gene expression via epigenetic reprogramming.T hus , it would be interesting to see whether the variation in nsp1 sequences across cor onaviruses r eflects the div ersity of nsp1-mediated epigenetic mechanisms.

Conclusions
Through millions of years of evolution, viruses and their hosts hav e been loc ked in an endless arms race , trying to sta y one step ahead of each other.This arms race takes many forms and dimensions, with each side employing a variety of strategies to gain an adv anta ge.Ho w e v er, viruses and their hosts differ fundamentally in one critical aspect of this arms race.Due to their compact genomes, viruses often lack the ability to encode dedicated proteins to perform specific functions, forcing them to r el y on multifunctional proteins .T he nsp1 protein of SARS-CoV-2 is an excellent example of how a single viral protein can perform a variety of functions to e v ade host immune defenses.To date, the most extensiv el y studied function of nsp1 is to shut off cellular mRNA translation and promote its degradation.Furthermor e, nsp1 bloc ks nuclear export of mRNA and inhibits the activation of IFN signaling.Ho w ever, new evidence suggests that nsp1 is functionally even more complex than previously thought.We now know that nsp1 promotes epigenetic silencing of antivir al imm une-r elated genes thr ough the G9a-mediated deposition of the r epr essiv e mark H3K9me2 on the c hr omatin.T his disco very not only adds another layer to our understanding of how nsp1 performs its multiple functions, but also raises new questions.A k e y open question is how exactly nsp1 induces H3K9me2-based heter oc hr omatin formation.It is particularly important to identify the host factors involved and determine whether nsp1 performs its epigenetic function dir ectl y in the nucleus or indir ectl y thr ough other proteins that shuttle between the cytoplasm and the nucleus.Another critical question is what determines the specificity of nsp1-mediated epigenetic silencing for imm une-r elated gene loci.Despite intriguing hints fr om inter actome studies, the abov e putativ e mec hanisms underl ying nsp1-mediated epigenetic silencing are, at best, speculative and require experimental validation.A better understanding of these mechanisms may lead to the de v elopment of nov el nsp1-specific inhibitors , thus pa ving the way for new treatments for SARS-CoV-2 as well as other circulating and emerging coronavirus infections.As discussed above, virus-induced epigenetic r epr ogr amming of host gene expression may contribute to the long-term effects of SARS-CoV-2 infection, also known as the "long CO VID syndrome." It would be interesting to know whether the nsp1-induced epigenetic changes play a role in the de v elopment of this heter ogeneous syndr ome with div erse clinical manifestations.

Figure 1 .
Figure 1.Specific interaction between nsp1 and the catalytic subunit of DNA pol ymer ase alpha (POLA1) may affect its epigenetic functions.(A) Domain map of POLA1 showing the nsp1-binding region and sequence motifs involved in epigenetic inheritance.Based on the diagram by Nasheuer and Onwubiko ( 2023 ).(B) The interaction between nsp1 and POLA1 may prevent regulatory proteins from accessing the flexible N-terminal region of POLA1.POLA1 is shown as a gray space-filling model.A cross-section of the catalytically inactive POLA1 exonuclease domain is shown in solid gray.The contact area between POLA1 and nsp1 is highlighted with the dashed line.Nsp1 is drawn as ribbons, with POLA1-interacting structural elements in purple and the rest in green.The dotted circle indicates a potential steric clash between nsp1 and binding partners of the flexible N-terminal region of POLA1.The schematic rendering of the nsp1-POLA1 complex is based on the published cryo-EM structure by Kilkenny et al. ( 2022 ).
Furthermor e, these mec hanisms may pr omote imm une e v asion in conjunction with other canonical nsp1-mediated mechanisms, suc h as tr anslational r epr ession, mRNA degr adation, and inhibition of mRNA export from the nucleus.Nuclear translocation of phosphorylated ST A T (pST A T) is an integral part of the canonical JAK/ST A T pathwa y, which pla ys a centr al r ole in the IFN-mediated antivir al r esponse.In the canonical pathway, unphosphorylated ST A T transcription factors (uST A Ts) ar e inactiv e in the cytoplasm and r equir e phosphorylation for acti vation and n uclear translocation.Ho w ever, a pool of uSTATs, including uST A T1 (Me yer et al. 2002 ), is also present in the n u-

Figure 2 .
Figure 2. Nsp1 promotes transcriptional and epigenetic silencing of imm une-r elated genes.(A) A model describing the role of nsp1 in ISG r epr ession.Nsp1 anta gonizes type I IFN signaling by inhibiting ST A T1 phosphorylation in the cytoplasm.As a result, ST A T1 is unable to form activated dimers with ST A T2, preventing them from translocating into the nucleus and inducing ISG expression.In addition, nsp1 promotes G9a-mediated deposition of the r epr essiv e c hr omatin mark H3K9me2 at imm une-r elated genomic loci through a yet unknown mechanism.The H3K9me2 mark is specifically recognized by HP1, promoting facultative heter oc hr omatin formation.Nuclear unphosphorylated ST A Ts (uST A Ts), such as uST A T3, may stabilize heterochromatin by specifically interacting with HP1.(B) All human ST A T transcription factors contain the HP1-binding motif PxVxL/I (highlighted in black).Shown is an amino acid sequence alignment of the regions surrounding the HP1-binding motif in se v en members of the human ST A T protein family.